PROJECT SUMMARY: Vaccination is one of the most successful public health interventions in human history. Despite the fact that many incurable and high-mortality diseases are fully controlled, and in some cases eliminated from human civilization, many other diseases remain elusive to vaccine interventions. Our ability to better understand how natural infectious pathogens trigger and control mammalian immunity by activating multiple immune-pathways and generating a combinatorial response, will greatly help the design and clinical translation of more effective vaccines against emerging infectious diseases, HIV/AIDS, cancer, etc. Most of our knowledge in stimulating vaccine adjuvants, molecules that stimulate the immune system to generate protective or therapeutic immunity against antigens, comes from pathogen/danger-associated molecular patterns (PAMPs/DAMPs) of viruses, bacteria, fungi or parasites. In natural infections, multiple adjuvants and Ag are carried inside a particle-like structure and innate immune cells experience adjuvant and antigens as a combination entity in single particles, which acts to localize and concentrate a set of synergistic stimulatory signals. Thus, we argue, that (a) in order to develop more efficacious vaccines we must understand the molecular mechanisms by which multiple adjuvants act on innate immune cells and in tandem, control adaptive immunity; and (b) the most prudent way of presenting combinations of adjuvants in vivo is through particulate carriers that mimic pathogens (Pathogen-like particles, PLPs). Variation of these carriers' properties (e.g. size, charge, composition) will affect how adjuvants interact with innate immune cells and modulate the resulting immune response; an aspect that likely occurs in natural infections as well. Our overarching hypotheses are that the combinatorial in vivo effects of two clinically-relevant adjuvants, CpG and Monophosphoryl Lipid A (MPLA), can be (a) precisely modulated by the mode of presentation (soluble, vs particulate carriers of different physical properties) and (b) better predicted by in vitro assays when delivered together as particulate carriers. To test these, we propose the following aims. Aim 1: Develop and characterize CpG/MPLA co-loaded pathogen-like-particle (PLP) adjuvants of various sizes and adjuvant- density. Aim 2: Investigate in-vitro, the molecular mechanisms involved in how various DC subsets respond to combination adjuvant formulations. Aim 3: Identify in vivo, the molecular mechanisms behind synergistic systemic immune-responses to combination adjuvant formulations. Collectively, the proposed studies will (a) greatly advance our understanding of molecular mechanisms that mediate potent priming of adaptive immunity by combination-adjuvants, (b) identify key physico-chemical parameters that control adjuvanticity of combination-adjuvants and PLPs, and (c) lead to the development of new platform technologies and reagents for combinatorial-adjuvant based human vaccines with immediate translational potential and clinical use.